Targeted delivery of BMPR2 mRNA attenuates pulmonary arterial hypertension by reversing pulmonary vascular remodeling

Figure 1. Schematic overview of the main contents. Our therapeutic development process encompasses three pivotal phases: 1) Formulation optimization: via three iterative cycles of DOE optimization—a systematic and statistically robust methodology for designing, executing, and analyzing experiments to identify critical variables, optimize formulation parameters, and establish quantitative relationships—we successfully refined the LNP composition from an initial four-component system (ionizable lipid, cholesterol, DOPE, and DMG-PEG) to an optimized three-component formulation (ionizable lipid, cholesterol, and DMG-PEG). This refinement resulted in a significant 35-fold improvement in pulmonary delivery efficiency in ICR mice using the A2-3O14 ionizable lipid. 2) Potential application in large animals: acknowledging interspecies variability, we conducted a systematic evaluation of ten representative ionizable lipids from our prior library (including A2-3O14) in SD rats utilizing the optimized formulation. This comparative analysis identified A5-3O14 as the most suitable ionizable lipid for rats. 3) Therapeutic validation: following comprehensive safety assessments, we evaluated A5-3O14 LNPs…

Figure 2. Formulation optimization of lung-targeting LNPs. (A) Schematic of formulation optimization: A2-3O14, cholesterol, helper lipid, and DMG-PEG were used to form LNPs for the encapsulation of Luc mRNA. The DOE approach was utilized to optimize the formulation with maximized pulmonary delivery efficiency. Optimization was conducted in ICR mice, resulting in a highly effective lung-targeted three-element formulation. n = 3. (B) Parameter setting for different rounds. The combined molar percentage of cholesterol, phospholipid, ionizable lipid, and PEG was fixed at 100%. CBS: sodium citrate buffer. (C) Particle size, PDI, and zeta potential of 22 LNPs in round A. (D) Factors with the most significant influence on particle size in round A. (E) Pulmonary luciferase expression efficiency of various LNPs in round B after intravenous injection to ICR mice with a dose of 0.1 mg/kg of Luc mRNA. Data are plotted as the mean ± SEM. (F) Factors with the greatest influence on the efficiency of Luc mRNA expression in the DOPE group of round B. (G) Pulmonary luciferase expression efficiency of various LNPs in round C after intravenous injection of 0.1 mg/kg of Luc mRNA to ICR mice. Data are plotted…

Figure 3. A5-3O14 LNPs exhibited high pulmonary delivery efficiency and biocompatibility in SD rats. (A) Leading ionizable lipids with pulmonary mRNA delivery capacity in ICR mice were evaluated in rats. The LNPs were formulated according to DSPC-C6 formulation and i.v. injected to rats at a dose of 0.1 mg/kg Luc mRNA. Presented here were the ex vivo multi-organ bioluminescence images of the three most efficient ionizable lipids in rats. (B) A statistical plot of the Luc mRNA expression efficiency in the lung for the three ionizable lipids in A (n = 6). (C) The Z-Ave and PDI of the top three LNPs in B. (D) TEM images of the A5-3O14 LNPs. The dot indicated by the black arrow represented mRNA. (E) BMPR2 expression in healthy rats after injecting varying doses (mg/kg) of BMPR2 mRNA LNPs for 6 h (n = 3). (F) The quantification of BMPR2 expression in E (fold change relative to β-actin). (G) BMPR2 expression in healthy rats after injecting varying doses (mg/kg) of Luc mRNA LNPs for 6 h (n = 3). (H) The quantification of BMPR2 expression in G (fold change relative to β-actin). (I) BMPR2 expression in 1-week-MCT-challenged rats that were treated with BMPR2 mRNA LNPs (0.2 mg/kg) or dexamethasone (1…

Figure 4. BMPR2 protein replacement therapy prevents the disease progression of MCT rats. (A) Schematic illustration of the experimental design. On Day 0, SD rats were given a vehicle injection (wild type control, n = 12) or treated with MCT (n = 48, 60 mg/kg, s.c.). MCT injected rats were randomly separated into four groups, including LNPH (n = 12), LNPL (n = 12), DEX (n = 12), and MCT CTRL (n = 12). (B–E) Echocardiography was performed to measure the ratio of PAT/PET in rats (n = 8–12 per group). B, PAT/PET on Day 7. C, PAT/PET on Day 13. D, PAT/PET on Day 22. E, Representative echocardiography images on Day 22. (F) Representative Western blot for BMPR2, phosphorylated SMAD1/5/9 (p-SMAD1/5/9), ID1 and β-actin in whole lung lysate (n = 3). (G) The quantification of BMPR2 expression in F (fold change relative to β-actin). (H) The quantification of p-SMAD1/5/9 expression in F (fold change relative to β-actin). (I) Representative hematoxylin and eosin (H&E) images of pulmonary arterioles. Scale bars, 20 μm. (J) The summarized data of (I). The pulmonary arteriole thickness (PA) was expressed as the ratio of wall area to total vessel area. (K) Statistical chart of the proportion of pulmonary…

Figure 5. BMPR2 protein replacement therapy prevents SU5416-hypoxia-induced PAH in rats. (A) Schematic illustration of the experimental design. On Day 0, SD rats were given vehicle injections and maintained in normoxia (WT CTRL, n = 10) or challenged with SU-5416 (20 mg/kg, s.c.) and exposing them to 3 weeks of hypoxia (10% O2) before normoxia (n = 30). SU-5416-hypoxia rats were randomly separated into three groups, including untreated group (SU CTRL, n = 10), group treated with 1 mg/kg dexamethasone (Dex, n = 10), and group treated with 0.1 mg/kg BMPR2 mRNA-LNP and 1 mg/kg dexamethasone (LNP, n = 10). The prevention treatment started from Day 11 and was treated every 5 days until Day 36, and the ultrasound monitoring was carried out on Days 17, 37 and 57. On Day 37, half of the mice from each group were sacrificed for therapeutic assessment after the ultrasound monitoring, while the remaining mice were kept in normoxia until Day 57 to evaluate the duration of therapeutic benefit. (B) Representative echocardiography images on Days 37 and 57. (C) PAT/PET on Day 17 (n = 9–10). (D) PAT/PET on Day 37 (n = 8–10). (E) PAT/PET on Day 57 (n = 5). (F) Fulton index on Day 37 (n = 3–5). (G) Fulton…